Process for producing plant storage organ with high production of recombinant protein and novel recombinant protein

ABSTRACT

The present invention provides a method for highly producing a recombinant protein in a plant storage organ and a GLP-1 derivative. The plant storage organ in which the recombinant protein is highly produced is obtained by transformation with the use of a vector which comprises a recombinant protein gene, a cytokinin-related gene, a drug-resistant gene and a removable DNA element, in which the cytokinin-related gene and the drug-resistant gene exist in the positions so that they can behave together with the DNA element, while the recombinant protein to be expressed in the plant storage organ exists in the position so that it would not behave together with the DNA element. The GLP-1 is produced by using the method, and a derivative having been stabilized against enzymatic digestion is further provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.10/550,624, which is the U.S. National Stage application ofPCT/JP04/04382, filed Mar. 26, 2004, which claims priority from Japaneseapplication Japan 2003-092827, filed Mar. 28, 2003.

TECHNICAL FIELD

The present invention relates to a method for producing a plant storageorgan in which a recombinant protein is highly produced and a novelderivative of human glucagon-like peptide-1 (GLP-1) which ispeptidase-resistant and the use thereof. Meanwhile, the “recombinantprotein” in the present invention encompasses “a recombinant peptide anda recombinant protein” (hereinafter referred to as “a recombinantprotein”).

BACKGROUND ART

Production of pharmaceuticals, clinical diagnostics and industrialmaterials using genetic engineering techniques has greatly contributedto the actual industrial world already, among which substance productionsystems are particularly widely utilized where cultured cells ofmicroorganisms or mammals are used as host cells. However, culture ofthese cells requires culture facilities or culture media in completelysterile environments. The further inevitable consumption of petroleumenergy causes high cost. In addition, mammalian cells cannot be used ashosts without involving the risk of contamination of viruses which areharmful to the human body.

Consequently, substance production systems using transformed plants havebeen developed as substance production systems with safety at low costinstead of substance production systems using cultured cells ofmicroorganisms or mammals. For instance, generation of trans formedplants producing a polymeric compound such as biodegradable polyester(e.g. Japanese Laid-Open Patent Application No. 2002-262886), a proteinsuch as a vaccine (e.g. G. Jaeger et al., Eur. J. Biochem. 259, 426,1999) and lactoferrin (D. Chong et al., Transgenic. Res. 9, 71, 2000),and a peptide such as enkephalin (Japanese-Laid Open Patent ApplicationNo. 2000-106890), have been reported so far.

With regard to transformed plants, production of a functional substancebeing beneficial to the human body in edible parts of the plants e.g.seeds of Glycine max or Oryza sativa, or vegetable leaves, allows theintended substance to be taken orally into the human body directlywithout an extraction process for them. Further, for seeds,refrigeration or transportation in a facility with a refrigeratingdevice is not required, because they can be steadily stored for a longtime at room temperature. In addition, even when the intended substanceis extracted, it can be easily purified, because, unlike leaves, thecontamination of phenolic substances seldom occurs with seeds.Accordingly, a seed has been regarded as an ideal organ to produce theintended genetic product, and generation of seeds which producedproteins such as glycinin (T. Katsube et al., Plant. Physiol. 120, 1063,1999), enzymes such as (1,3-1,4)-β-glucanase (H. Horvath et al., Proc.Nathl. Acad. Sci. USA., 97, 1914, 2000), and peptides such as enkephalin(D. Chong et al., Transgenic. Res., 9, 71, 2000) have been reported sofar.

However, although substance production systems by transformed plantshave the above superior properties, their production efficiency isinferior to that of culture systems using microorganisms or mammaliancells which are the current mainstream, particularly, the productionefficiency by plant storage organs is low. In order to solve thisproblem, measures are variously being devised to enhance the ability toproduce substances in transformed plants. For instance, in order toimprove the ability to produce substances in one of the storage organs,i.e. a seed, from the point of view of enhancing the expression of theintroduced intended gene and accumulation of a gene product, studiesregarding utilization of a promoter of a plant storage protein expressedintensively in seeds (e.g. T. Katube et al., Plant. Physiol., 120, 1063,1999), concomitant use of this promoter and a transcription factor whichacts on the promoter to enhance expression (e.g. D. Yang et al., Proc,Nathl. Acad. Sci. USA., 98, 11438, 2001), insertion of 5′ enduntranslated region (e.g. Japanese Laid-Open Patent Application No.2002-58492), optimization of C+G content in a gene (H. Horvath et al.,Proc. Nathl. Acad. Sci. USA., 97, 1914, 2000), addition of transductionsignals to an endoplasmic reticulum (Japanese Laid-Open PatentApplication No. 2000-504567), and so on have been performedenergetically. It is also reported that the production amount of aforeign gene product in the seed was increased by us ing a mutantdeficient in a seed storage protein as a plant into which a foreign geneis introduced (Japanese Laid-Open Patent Application No. 2002-58492).However, these improvements have not provided enough substanceproduction ability in seeds, so that development of a novel procedurehas been longed for.

On the other hand, a GLP-1 (glucagon-like peptide-1) is known as ahormone which is secreted from a digestive tract by food intake and actson the pancreas to stimulate glucose-dependent insulin secretion. InType 2 diabetic patients, it is reported that responsiveness to thisGLP-1 is maintained, while the production of GLP-1 is impaired. It isexpected that development of a GLP-1 agent will lead to the applicationof the agent to a therapeutic agent for diabetes as an insulin secretionpromoter to compensate for the lack of the GLP-1. However, the activesubstance of the GLP-1 is a polypeptide of the GLP-1 (7-36) amide or theGLP-1 (7-37), which are digested and degraded by a digestive enzyme inthe gastrointestinal tract and is not absorbed sufficiently when theGLP-1 is taken orally. Therefore, in the present state, intravenousinjection and subcutaneous injection are attempted in clinical practice.Moreover, it is also reported that the GLP-1 is also subjected todegradation by a dipeptidylpeptidase IV (DPP-IV) which exists in bloodand tissues, so the active half-life of the GLP-1 is as short as 1-2min, and GLP-1 is easily excreted from the kidney, so its half-life inblood is within 5 min, all of which prevents the clinical application ofGLP-1.

Hence, a GLP-1 derivative with a long half-life which is not easilydegraded has been developed. For instance, the following are included: aderivative substituted at the 8^(th) amino acid position (diabetologia41, 271-278, 1998, Biochem 40, 2860-2869, 2001), an amino acid modulatorat N- and C-terminals (WO9808871 etc.), a derivative in which Arg issubstituted at its 34^(th) position and its 26^(th) position of Lys isintroduced with a lipophilic group (WO0007617), and a derivativeobtained by amino acid substitution covering all over the sequence(WO9943705 and WO9111457). Further, development of a sustained-releaseinjection preparation which is subcutaneously absorbed slowly, ordevelopment of an injection preparation with synthetic Exendin-4 havinga GLP-1 like agonist activity and derived from lizard whose half-life inblood is long, have been performed. However, as they are injectionpreparations, considering the burden to patients, a novel GLP-1derivative administered via an alternative route other than injectionhas been longed for.

The object of the present invention is to provide a method for producinga plant storage organ in which a recombinant protein is highly produced,a plant storage organ in which the recombinant protein produced by themethod is highly produced, and a novel derivative of a humanglucagon-like peptide-1 (GLP-1) which is peptidase-resistant and the usethereof.

In order to enhance substance production in a storage organ of atransformed plant, various attempts have been performed as describedabove. However, in order for a plant storage organ to sufficientlyfunction in vivo to produce a recombinant protein useful as apharmaceutical as food, it is necessary to develop a method forproducing a plant storage organ in which the recombinant protein is morehighly produced. In the meantime, when the recombinant protein isextracted from plants and processed as a pharmaceutical or functionalfood, it is important that the recombinant protein is highly produced inthese storage organs on the cost front. Therefore, one of the objects ofthe present invention is to provide a novel method for producing astorage organ in which the recombinant protein is highly produced intransformed plants.

Meanwhile, when a GLP-1 is selected as a recombinant protein which ishighly produced in a plant storage organ by said method, a therapeuticeffect for diabetes can be expected by merely taking fruits, rice, andso on as normal diet. However, as mentioned above, since this nativeGLP-1 is digested and degraded by the digestive enzyme in thegastrointestinal tract, it cannot be orally administered stably, thereis no efficient method for administration except injection in thecurrent status. It can be thought that if the GLP-1 can be passedthrough the stomach without being digested using some method, it isabsorbed in the small intestine. However, the GLP-1 must exist as asimple substance when it is absorbed. In that time, a native GLP-1 wouldlose activity by degradation by an enzyme such as trypsin.

Moreover, as the native GLP-1 is continuously degraded bydipeptidylpeptidase IV even after absorption, a sustained effect cannotbe expected. Accordingly, in order to obtain a pharmaceutical effectfrom oral administration of the GLP-1, it is necessary to design byamino acid substitution a GLP-1 derivative which is not easily degradedwith trypsin or dipeptidylpeptidase IV and which has sustained activity.

Therefore, one of the other objects of the present invention is toprovide a novel GLP-1 derivative which is resistant to a digestiveenzyme such as trypsin and can be administered orally, more preferably,a novel GLP-1 derivative which is resistant to dipeptidylpeptidase IV aswell. To accomplish this object, it is required to obtain a GLP-1derivative which is absorbed when taken as food, and which shows apharmaceutical effect.

DISCLOSURE OF THE INVENTION

The present inventors have made a keen study concerning a method forproducing a storage organ in which a recombinant protein is highlyproduced in a transformed plant, as a result they found that a storageorgan in which a recombinant protein is highly produced in a transformed plant can be produced, through the following steps: constructinga vector which comprises a recombinant protein gene to be expressed in aplant storage organ, a cytokinin-related gene, a drug-resistant gene,and a removable DNA element, where the cytokinin-related gene and thedrug-resistant gene exist in the positions so that they can behavetogether with the removable DNA element, while the recombinant proteingene to be expressed in the plant storage organ exists in the positionso that it would not behave together with the removable DNA element,introducing the vector into cells, redifferentiating a transformant fromthe plant cell into which the gene is introduced, and obtaining astorage organ from the redifferentiated transformant. The presentinvention has been thus completed.

The present invention is to apply the method for producing a plantstorage organ in which a recombinant protein is highly produced to aGLP-1 known as a hormone stimulating glucose-dependent insulin secretionand to generate a GLP-1 derivative to provide the GLP-1 derivative whichis not digested or degraded by a digestive enzyme and so on, and furtherwhich is stable in blood plasma. In other words, the present inventorsfound that a GLP-1 derivative in which glutamine and asparagine oraspartic acid respectively are substituted at the 26^(th) and 34^(th)positions; in a peptide comprising GLP-1 (7-36) or its amino acidsequence in which one or a few amino acids are deleted, substitutedand/or added and having GLP-1 activity, maintains activity at the samelevel as the native GLP-1, is resistant to the digestive enzyme such astrypsin, and can be absorbed from the small intestine. Further, thepresent inventors found that the GLP-1 derivative is resistant todipeptidylpeptidase IV and is stable also in the blood plasma bysubstituting serine or glycine for alanine at the 8^(th) position, andthus completed the present invention. In addition, the peptide isdegraded by pepsin in the stomach when it is orally administered, so itwas conventionally impossible to administer the peptide orally. Byproducing the peptide in the plant storage organ of the presentinvention, however, pepsin-resistance can be obtained so that oraladministration can be performed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows preparation of pTL7 to pGlbGLP in the scheme for preparingpGlbGLP130Hm used in the Examples of the present invention.

FIG. 2 shows preparation of pUC18 and pNPI130 to pNPI130PUC in thescheme for preparing pGlbGLP130Hm used in the Examples of the presentinvention.

FIG. 3 shows preparation of pNPI140 and pNPI130PUC to pNPI130Hm in thescheme for preparing pGlbGLP130Hm used in the Examples of the presentinvention.

FIG. 4 shows preparation of pG1bGLP and pNPI130Hm to pG1bGLP130Hm aswell as a restriction map of pGlbGLP130Hm used in the Examples of thepresent invention.

FIG. 5 shows accumulation level of the GLP-1 derivative fusion-proteinin ripe seeds of Oryza sativa obtained in Example 1 and ComparativeExample 1 in the Examples of the present invention.

FIG. 6 shows a restriction map of the conventional vector pGlbGLP-Hm inthe Examples of the present invention.

FIG. 7 shows the measured result of cyclic AMP production activity ofGLP-1 (7-36 amide) (native GLP-1) in the Comparative Production Example1, [Ser⁸]-GLP-1 (7-36 amide) in the Comparative Production Example 2,[Gly⁸]-GLP-1 (7-36 amide) in the Comparative Production Example 3, and[Gln²⁶, Asn³⁴]-GLP-1 (7-36 amide) in the Production Example 1 accordingto the method shown in Example 2 in the Examples of the presentinvention.

FIG. 8 shows comparison of the concentration dependency of cyclic AMPproduction activity between trypsin treated [Gln²⁶, Asn³⁴]-GLP-1 (7-36amide) and untreated [Gln²⁶, Asn³⁴]-GLP-1 (7-36 amide) after treatingwith trypsin according to the method shown in Example 3 in the Examplesof the present invention.

FIG. 9 shows comparison of the stability to pepsin using a GLP-1derivative derived from polished rice of ripe seeds of Oryza sativaobtained in Example 1 and the powder thereof, GLP-1 (7-36 amide) (nativeGLP-1) in the Comparative Production Example 1, [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36) in the Production Example 2, and [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1 (7-36) in the Production Example 3 according to the methodshown in Example 4 in Examples of the present invention.

FIG. 10 shows the relationship between trypsin treatment time and cyclicAMP production activity of the extracted fraction after extracting[Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) as fusion protein from ripe seeds ofOryza sativa according to the method shown in Example 5 in the Examplesof the present invention.

FIG. 11 shows the comparison of the trypsin resistance using GLP-1 (7-36amide) (native GLP-1) in the Comparative Production Example 1, [Ser⁸,Gln²⁶, Asp³⁴]-GLP-1 (7-36) in the Production Example 2, and [Ser^(a),Gln²⁶, Asn³⁴]-GLP-1 (7-36) in the Production Example 3 according to themethod shown in Example 6 in the Examples of the present invention.

FIG. 12 shows the comparison of the DPP-IV resistance using GLP-1 (7-36amide) (native GLP-1) in the Comparative Production Example 1, [Ser⁸,Gln²⁶, Asp³⁴]-GLP-1 (7-36) in the Production Example 2, and [Ser⁸,Gln²⁶, Asn³⁴]-GLP-1 (7-36) in the Production Example 3 according to themethod shown in Example 7 in the Examples of the present invention.

FIG. 13 shows the comparison of the insulin secretion-promoting activityusing GLP-1 (7-36) (native GLP-1 amide) in the Comparative ProductionExample 1, [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) in the Production Example2, and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) in the Production Example 3according to the method shown in Example 8 in the Examples of thepresent invention.

FIG. 14 shows the comparison of the hypoglycemic effect in oral glucosetolerance test with mice using GLP-1 (7-36 amide) (native GLP-1) in theComparative Production Example 1, [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) inthe Production Example 2, and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) in theProduction Example 3 according to the method shown in Example 9 in theExamples of the present invention, and shows an area under the curve ofthe graph indicating variation of blood glucose level from 0 to 120 minin FIG. 15 as blood glucose level variation.

FIG. 15 shows comparison of the hypoglycemic effect in oral glucosetolerance test with mice using GLP-1 (7-36 amide) (native GLP-1) in theComparative Production Example 1, [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) inthe Production Example 2, and [Ser⁸, Gln²⁸, Asn³⁴]-GLP-1 (7-36) in theProduction Example 3 according to the method shown in Example 9 in theExamples of the present invention, and shows the change of blood glucoselevel from 0 to 120 min.

BEST MODE OF CARRYING OUT THE INVENTION

[Production of a Plant Storage Organ in which Recombinant Protein isHighly Produced]

The present invention is a method for producing a plant storage organ inwhich a recombinant protein is highly produced, comprising the followingsteps of (A), (B), and (C): (A) constructing a vector which comprises arecombinant protein gene to be expressed in a plant storage organ, acytokinin-related gene, a drug-resistant gene, and a removable DNAelement, where the cytokinin-related gene and the drug-resistant geneexist in the positions so that they can behave together with theremovable DNA element, while the recombinant protein gene to beexpressed in the plant storage organ exists in the position so that itwould not behave together with the removable DNA element, andintroducing the vector into cells, (B) redifferentiating thetransformant by culturing the plant cells into which the vector isintroduced by said step (A) in a drug-supplemented medium and adrug-free medium, and (C) obtaining a plant storage organ from thetransformant redifferentiated in said step (B). The present inventionwill be described in detail below.

(Subject Plants)

With regard to subject plants used in the production of the plantstorage organ in the present invention, it is not specifically limitedas long as a storage organ is formed in the plant, but as dicotyledon,Nicotiana tabacum, Brassica rapa var. nippo-oleifera, and Glycine max,while as monocotyledon grains such as Oryza sativa, Zea mays, Hordeumvulgare, and Triticum aestivum, and Asparagus officinalis can berepresented. Further, with regard to a plant storage organ in which therecombinant protein is highly produced in the present invention, it isnot specifically limited, but a fruit, a tuberous root, a tuber, a seed,and the like can be represented.

(Genes to be Used)

Genes to be used in the present invention can be obtained by cloning ofcDNA or genomic DNA. When the DNA sequence has been elucidated inadvance, it may be obtained by chemosynthesis. Further, though the DNAsequence has not been elucidated, if the amino acid sequence has beenrevealed, DNA sequence deduced from the amino acid sequence can beproduced by chemosynthesis.

In the present invention, according to the need, the sequence of thepromoter and/or the terminator necessary for gene expression is linkedto the signal sequence to transfer the gene product to a storage organefficiently and used as a gene. These sequences of the promoter, theterminator, and the signal can be used without limitation as long asthey function in plants. As for this type of promoter, e.g. 35S promoterof cauliflower mosaic virus (J. T. Odell et al., Nature (London), 313,810, 1985), the promoter of nopaline synthase (W. H. R. Langridge etal., Plant Cell Rep., 4, 355, 1985) and the like can be used. Further,use of an inductive promoter controls gene expression.

A number of such inductive promoters have been known so far. Forinstance, as for promoters which are induced by responding to chemicalsubstances, the following are known: a promoter of aglutathione-S-transferase I gene (Japanese Laid-Open Patent ApplicationNo. 5-268965), a promoter of a glutathione-S-transferase II gene(International Publication WO93/01294), a Tet-repressor fusioncauliflower mosaic virus 35S promoter (C. Gatz et al., Mol. Gen. Genet.,227, 229, 1991), a Lac operator/repressor promoter (R. J. Wilde et al.,The EMBO Journal, 11, 1251, 1992), an alcR/alcA promoter (InternationalPublication WO94/03619), a glucocorticoid promoter (Aoyama, PROTEIN,NUCLEIC ACID AND ENZYME, 41: 2559, 1996), and a par promoter (T. Sakaiet al., Plant Cell Physiol., 37, 906, 1996). As promoters which areinduced by responding to light, followings are known: a promoter of aribulose diphosphate carboxylase small subunit gene (rbcS) (R. Fluhr etal., Proc. Natl. Acad. Sci. USA, 83, 2358, 1986), a promoter of afructose-1,6-bisphosphatase gene (Japanese Patent Publication No.7-501921), a promoter of a light-harvesting chlorophyll a/b bindingprotein gene (Japanese Laid-Open Patent Application No. 5-89) and thelike. Other than above, promoters which are induced by responding tovarious external environments such as injury, temperature and the like,are known.

As for a promoter, for the recombinant protein gene of the presentinvention, an inductive promoter and a promoter showing constantexpression such as a 35S promoter can be used as described above, but itis particularly desirable to use a promoter specific to the plantstorage organ, since the expression of the promoter specific to theplant storage organ is guaranteed to be expressed in the plant storageorgan in which the recombinant protein gene is attempted to be produced.Thus, the promoter which promotes specific expression in certain tissuesor organs in plants is also known widely to those of skill in the art.For instance, in the present invention, as for the promoter, a promoterof a globulin gene (M. Nakase et al., Plant Mol. Biol., 33, 513, 1997),a promoter of a glutelin gene (F. Takaiwa et al., Plant Mol. Biol., 17,875, 1991), and the like can be used, which are promoters of seedstorage protein genes which express foreign genes in seeds of Oryzasativa. Further, promoters of seed storage protein genes of major cropssuch as a promoter of a glycinin gene, a promoter of a glutelin gene (J.Rodin et al., plant Mol. Biol., 20, 559, 1992), and the like can also beused, which are promoters expressing foreign genes in seeds of Fabaceaecrops such as Phaseolus vulgaris, Vicia faba, Pisum sativum, and so onand seeds of oil seed crops such as Arachis hypogaea, Sesamum indicum,Brassica rapa var. nippo-oleifera, Gossypium arboreum, Helianthusannuus, Carthamus tinctorius L., and so on.

On the other hand, in the present invention, terminators of the plantgenes registered in DNA data base including a terminator of a nopalinesynthase (A. Depicker et al., J. Mol. Appl. Gen., 1, 561, 1982), and aterminator of octopine synthase (J. Gielen et al., EMBO J., 3, 835,1984) can be selected variously and used.

In the present invention, the recombinant protein gene which can beintroduced into a plant may not only be a gene encoding a functional ormedical peptide capable of contributing to the health of humans andanimals such as livestock, but also may be a gene encoding an optionalpeptide or protein whose function is unknown. For instance, in theExamples of the present invention, the gene encoding a GLP-1 derivativewas introduced into a plant and the GLP-1 derivative was produced in theplant seed, but the recombinant protein which can be produced in theplant storage organ according to the procedure of the present inventionis not limited to the GLP-1 or the derivative thereof, and variouspeptides and proteins such as various peptides or proteins having beenused or developed as pharmaceuticals already (S. Josephson and R.Bishop, TIBTECH, 6, 218, 1998), a recently found hypocholesterolemicpeptide (e.g. Japanese Laid-Open Patent Application No. 2001-114800), aT-cell epitope peptide of tick or pollen antigen (e.g. U.S. Pat. No.6,268,491, Japanese Laid-Open Patent Application Nos. 10-7700,10-259198, 10-506877, 11-92497, and 2000-327699), and the like, can beproduced in a plant storage organ according to the procedure of thepresent invention.

Additionally, these peptides may be produced with suitable modificationaccording to the nature and the object of the invention. That is, asexemplifying the GLP-1, other than the GLP-1, the present invention canbe applied to a peptide comprising a GLP-1 (7-36) or its sequence inwhich one or a few amino acids are deleted, substituted and/or added andhaving GLP-1 activity, or a GLP-1 derivative comprising an amino acidsequence in which glutamine and asparagine or aspartic acid arerespectively substituted at the 26^(th) and 34^(th) positions of thepeptide. Further, the present invention can also be applied to a GLP-1derivative whose peptide, which comprises a GLP-1 (7-36) or its sequencein which one or a few amino acids are deleted, substituted and/or addedand which has GLP-1 activity, is GLP-1 (7-36), GLP-1 (7-37), orC-terminal amide of GLP-1 (7-36) or GLP-1 (7-37). Moreover, the presentinvention can also be applied to a GLP-1 derivative in which serine orglycine is substituted at the 8^(th) positions of these GLP-1derivatives, and the GLP-1 derivative shown in SEQ ID NO: 2 in thesequence listing.

(Construction of Recombinant Protein Gene to be Introduced)

In the present invention, a fusion gene produced by: inserting the gene(DNA sequence) encoding these recombinant proteins into the geneticsequence encoding the variable region, which is not negatively affectedon accumulation or the like of the protein, in the protein gene such asa seed storage protein originally expressed in the plant storage organin which recombinant protein is to be highly produced according to theprocedure of the present invention, or by substituting for the gene, canbe used. For instance in the Examples, the gene encoding the above GLP-1derivative was inserted into the protein variable region in a globulingene, and used as a fusion gene. At the same time, by aligning an enzymefragmentation sequence at the boundary between the recombinant proteingene and the reserve protein gene which is originally expressed in theplant storage organ where the recombinant protein gene is inserted orsubstituted for, the object recombinant protein can be cleaved andpurified after the expression product of the fusion gene is extractedand treated with the enzyme. Further, aligning the cleaved sequence by adigestive enzyme such as trypsin there, the object peptide or protein iscleaved in the small intestine and absorbed into the body after theplant storage organs such as seeds in which the recombinant protein ishighly produced by the procedure of the present invention are taken asfood, which results in various physiological functions being exerted.

Meanwhile, the seed storage protein is a protein stored mainly in aseed, and has an important function as a nutrient necessary forgermination (Science of the Rice Plant vol. 3, Rural CultureAssociation). The type of the seed storage protein gene which can beused in the present invention is not specifically limited to, forinstance, a gene such as of globulin, glutelin, and prolamin of Oryzasativa, and 2s albumin of Arabidopsis thaliana (Japanese Laid-OpenPatent Application No. 2000-106890) can be used. Further, the insertionposition of the recombinant protein gene is not specifically limited aslong as it is in a variable region which does not change the property ofthe protein which is originally encoded by the seed storage proteingene. For instance, in the Examples of the present invention, a geneencoding the GLP-1 derivative was inserted into the position whichencodes the 109^(th) amino acid position of rice globulin.

(Construction of the Introducing Vector)

In the present invention, a gene is introduced into a plant by thevector constructed so that the cytokinin-related gene and thedrug-resistant gene exist in the positions so that they can behavetogether with the removable DNA element, while said recombinant proteingene exists in the position so that it would not behave together withthe removable DNA element.

Here, the cytokinin-related gene is referred to as a gene involved inproduction of cytokinin and so on which has functions causing promotionof cell division in a plant, differentiation of a definite bud or anadventitious bud from the plant tissue, or the like.

As for the cytokinin-related gene, other than an ipt gene derived fromAgrobacterium tumefaciens (hereinafter abbreviated as A. tumefaciens)(A. C. Smigocki, L. D. Owens, Proc. Natl. Acad. Sci. USA 85, 5131,1988), an ipt gene derived from Rhodococcus, a cytokinin synthase genederived from Arabidopsis thaliana, and a cytokinin synthase gene such asa ptz gene derived from Pseudomonas, any cytokinin-related genes of aβ-glucuronidase gene derived from E. coli which is a gene activatinginactive cytokinin (Morten Joersbo and Finn T. Okkels, Plant CellReports 16, 219-221, 1996), and a CKI1 gene derived from Arabidopsisthaliana thought as a cytokinin-receptor gene (Kakimoto T. Science 274,982-985, 1996), can be used in the present invention.

Further, in the present invention, the drug-resistant gene is referredto as a gene which confers antibiotic resistance or pesticide resistanceto the plant cell into which the drug-resistant gene is introduced. Asfor the antibiotic-resistant gene, a hygromycin-resistant gene (HPT: ahygromycin phosphorylated enzyme gene), a kanamycin-resistant gene(NPTII: a neomycin phosphorylated enzyme gene), and the like can be usedfor example, while as for the pesticide-resistant gene, asulfonylurea-resistant gene (ALS; an acetolactate synthase gene) and thelike can be used.

The removable DNA element is referred to as a DNA sequence which has anability to move from the chromosomal DNA or the like where it exists andfunctions. In plants, what is called a transposon existing onchromosomal DNA has been known as one of these elements, whosestructure, function, and behavior have been almost elucidated. In otherwords, in order for a transposon to function, two constituents arerequired in principle: an enzyme which is expressed from the geneexisting therein and catalyzes movement and transfer of the enzyme perse (transferase), and the DNA sequence that also exists in the terminalregion therein and to which the transferase binds and on which it acts.By these functions, a transposon moves from the chromosomal DNA on whichit exists, and it generally transfers to the new position on the DNA,however, there is a case that the transposon loses its function withouttransferring and disappears at a constant rate, therefore suchtransferring error of the transposon is used in the present invention.

Meanwhile, with regard to the transposon, other than such autonomoustransposon which possesses two constituents of transferase and DNAbinding sequence and can move autonomously from the chromosome on whichit exists by the action resulted from binding of the transferase whichis expressed from the inside of the transposon to the DNA sequenceexisting at the terminal region, and then it can transfer, there is alsoa type called a nonautonomous transposon. This nonautonomous transposonis referred to as one which possesses the DNA sequence at the terminalregion to which the transferase binds and on which it acts, though itcannot move autonomously from the chromosome due to lack of transferaseexpression caused by mutation of the transferase gene therein. However,when the transferase is provided from the autonomous transposon or thetransferase gene exists independently of it, the nonautonomoustransferase shows behavior similar to that of the autonomous transposon.

With regard to the autonomous transposon, there is Ac, Spm, and the likewhich are isolated from Zea mays (A. Gieri and H. Saedler, Plant Mol.Biol., 19, 39, 1992). Especially, Ac can be obtained by cleaving a wx-m7gene locus in the chromosome of Zea mays with a restriction enzyme Sau3A(U. Behrens et al., Mol. Gen. Genet. 194, 346, 1984), it is theautonomous transposon which is the most analyzed plant transposon andits DNA sequence has already been elucidated (M. Muller-Neumann et al.,Mol. Gen. Genet., 198, 19, 1984), and those skilled in the art canobtain it easily, therefore, it is suitable for the DNA element used inthe present invention. Further, with regard to the nonautonomoustransposon, including Ds and dSpm in which the internal regions of Acand Spm are deleted, respectively (H.-P. Doring and P. Starlinger, Ann.Rev. Genet. 20, 175, 1986), nonautonomous transposons isolated from avariety of plants such as Antirrhium majus, Pharbitis nil, etc. otherthan Zea mays (e.g. Y. Inagaki et al., Plant Cell, 6, 375, 1994), areknown.

Incidentally, such a transposon has been known from many examples that,even if it is introduced into the plant chromosome whose species isdifferent from the one from which it is derived, it exerts its abilityto move and transfer (e.g. B. Baker et al., Proc. Natl. Acad. Sci. USA,83. 4844, 1986). Meanwhile, in the present invention, either anautonomous or nonautonomous transposon can be used. When thenonautonomous transposon is used, the transferase gene obtained from theautonomous transposon or synthesized and so on is required to beintroduced in addition to the nonautonomous transposon, in such a case,it may be introduced by integrating with this nonautonomous transposoninto the vector of the present invention, or they may be introducedcompletely independently.

Further, as a removable DNA element existing in other than plants, onesderived from a site-specific recombination system are known. Thesite-specific recombination system comprises two constituents: arecombination site (which is equal to the removable DNA element of thepresent invention) having a characteristic DNA sequence, and an enzymewhich specifically binds to the DNA sequences and catalyzesrecombination between the sequences when there are two or more of thesequences. The DNA element shows activity when the DNA sequences existat two positions at a regular interval in the same direction on the sameDNA molecule, and the region between the sequences is removed from thisDNA molecule (a plasmid, a chromosome or the like), while when thesequences exist at two positions in the opposite direction, the regionis inverted. In the present invention, the moving action of the formeris utilized. Meanwhile, a gene encoding a recombinant enzyme does notnecessarily exist on the DNA molecule the same as that of therecombination site; it is known that it can cause movement and inversionbetween the DNA sequences as long as it only exists and expresses in thesame cell (N. L. Craig, Annu. Rev. Genet., 22, 77, 1998).

Currently, a Cre/lox system, a R/RS system, a FLP system, a cer system,a fim system, and so on isolated from microorganisms such as a phage,bacteria (e.g. E. coli), and yeast are known as a site-specificrecombination system (general statement in N. L. Craig, Annu. Rev.Genet., 22, 17, 1998), although it has not been confirmed whether thesite-specific recombination system exists in higher organisms. However,even when the site-specific recombination system is introduced into thespecies of organism different from the species such as plant from whichit is derived, it has been revealed that the site-specific recombinationsystem isolated from these microorganisms behaves in the same manner asit does in the organism from which it is originally derived, as theCre/lox system derived from P1 phage is used for the transgenic vectorfor introducing into plants in International Publication WO93/01283.Incidentally, in one Example of the present invention, the R/Rs system(H. Matsuzaki et al., J. Bacteriology, 172, 610, 1990), thesite-specific recombination system of yeast (Zygosaccharomyces rouxii),was used by inserting a recombinant enzyme between the recombinationsites. It has already been reported that the R/Rs system also maintainsthe original function in higher plants (H. Onouchi et al., Nucleic AcidRes., 19, 6373, 1991).

In the present invention, there is no limitation on the position toinsert the cytokinin-related gene and the drug-resistant gene into, aslong as it is the position where they can move with the removable DNAelement. For instance, when the autonomous transposon is used as aremovable DNA element, it can be inserted into the position which doesnot affect removal of transposon at the upstream from the promoterregion of a transferase gene and the downstream from the terminal regionto which the transferase gene binds. When the R/Rs system is used, itcan be inserted into any position, as long as it is the position whichdoes not inhibit the expression of the recombinant enzyme, and is in theregion between the recombination sites.

(Introduction of the Constructed Vectors into Plant Cells)

In the present invention, the constructed vector is introduced intoplant cells. With regard to the plants into which the vector isintroduced, as described in the above (subject plants) section, it isnot specifically limited as long as it is a plant forming a storageorgan, but grains such as Oryza sativa, Zea mays, Hordeum vulgare, andTriticum aestivum, and Asparagus officinalis as monocotyledon, andNicotiana tabacum, Brassica rapa. nippo-oleifera, and Glycine max asdicotyledon, can be exemplified as representative plants. In addition,the constructed vector can be introduced into a plant cell by usingknown methods. Besides the method using genus agrobacterium, any knownmethod such as electroporation method, polyethyleneglycol method, andparticle gun method can be used, and it is not specifically limited.

When the recombinant protein gene is introduced into Oryza sativa byusing the method of the present invention, for example, the methoddescribed in Japanese Patent No. 3141084 is preferably used. Here, withregard to the plating medium for plating a Oryza sativa seed andgerminating it, for instance an N6C12 medium (N6 inorganic salts andvitamins (Chu C. C., 1978, Proc. Symp., Plant Tissue Culture, SciencePress Beijing, 43-50), 30 g/L sucrose, 2.8 g/L proline, 0.3 g/L casaminoacid, 1 mg/L 2,4-D, 4 g/L GelRite) can be used. However, the mediumcomposition is not specifically limited to the above mentioned one, andthe present invention can also be carried out by modifying the type orconcentration of the composition.

(Redifferentiation of the Transformant)

When the transformant from the plant cell or tissue introduced with therecombinant protein gene is redifferentiated, the cell or tissueintroduced with the gene may be cultured by using the known method in adrug-supplemented medium and a drug-free medium. Meanwhile, thetransformant in the present invention is referred to as a plant tissuesuch as a definite bud, an adventitious bud, and an adventitious root ora seedling plant.

For instance, for obtaining a transformant by introducing therecombinant protein gene into Oryza sativa according to the presentinvention, the transformant can be obtained as a bud or a seedling plantby the following procedures: cleaving the scutellum tissue of Oryzasativa out of the geminated seed introduced with the gene according tothe method described in Japanese Patent No. 3141084 by using the vectorconstructed as mentioned above, culturing the scutellum tissue for oneweek in a medium such as a drug-supplemented medium N6C12TCH25 medium(N6 inorganic salts and vitamins, 30 g/L sucrose, 2.8 g/Lproline, 0.3g/L casamino acid, 2 mg/L 2,4-D, 500 mg/L carbenicillin, 25 mg/Lhygromycin, and 4 g/L GelRite), further culturing it for one week in adrug-supplemented medium N6C14TCH25 medium (N6 inorganic salts andvitamins, 30 g/L sucrose, 2.8 g/L proline, 0.3 g/L casamino acid, 4 mg/L2,4-D, 500 mg/L carbenicillin, 25 mg/L hygromycin, and 4 g/L GelRite),and then culturing it in a drug-free medium MSRC medium (MS inorganicsalts and vitamins (Murashige, T. and Skoog, F., 1962, Physiol. Plant.,15, 473), 30 g/L sucrose, 30 g/L sorbitol, 2 g/L casamino acid, 500 mg/Lcarbenicillin, and 4 g/L GelRite). The culture conditions exemplifiedabove are not absolute requirements, therefore, the type orconcentration of the medium composition can be modified, various planthormones or agents can be added to the medium, and the culture periodcan be modified as needed.

Meanwhile, with regard to the drug-supplemented medium, a medium towhich the drug suitable for the drug-resistant gene integrated into thevector constructed as described above and used for gene introduction isadded may be used. For example, a medium to which hygromicin, kanamycin,and sulfonylurea pesticide are added may be used when ahygromycin-resistant gene, a kanamycin-resistant gene, and asulfonylurea-resistant gene are integrated into this vector,respectively.

(Obtaining a Plant Storage Organ)

In the present invention, a plant storage organ may be obtained byredifferentiating the transformant as described above from the plantcell or organ into which the recombinant protein gene is introduced, andthen growing the transformant with the use of the known method. Forinstance, when the transformant is an adventitious bud, a storage organsuch as a plant maturing seed in which the recombinant protein is highlyproduced may be harvested after performing rhizogenesis treatment toregenerate the plant individual and growing the plant individual tofructify, according to the known method. Meanwhile, rhizogenesis of anadventitious root can be performed by a method such as incorporating anadventitious bud into an MS agar medium. In addition, when thetransformant is obtained as a seedling plant, the transformant plant canbe obtained without performing rhizogenesis treatment or the like.Further, when a tuberous root, a tuber, or the like is used as a plantstorage organ, it also can be obtained by differentiating these tissuesby known means from the obtained transformant without going through thegeneration process of the plant individual.

(Production of the GLP-1s)

In the present invention, GLP-1s are provided by using the method forproducing the plant storage organ in which the recombinant protein ishighly produced of the present invention. The GLP-1 is known as ahormone stimulating glucose-dependent insulin secretion, while GLP-1(7-36) is a peptide having a sequence shown byHis-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg(SEQ ID NO: 7). In the present invention, a gene encoding the amino acidsequence of the GLP-1 (7-36), or a gene encoding a peptide whichcomprises the sequence in which one or a few amino acids are deleted,substituted and/or added in the amino acid sequence of GLP-1 (7-36) andhas GLP-1 activity, is integrated as the gene expressing the aboverecombinant protein into the vector constructed in the present inventionto express the gene and produce GLP-1s.

(GLP-1 Derivative)

The Present Invention is to Provide the Method for producing the GLP-1sas described above and to generate a GLP-1 derivative which is notdigested or degraded by a digestive enzyme and so on, and further whichis stable also in blood plasma. The derivative of the present inventionwas modified so that it can be absorbed from the small intestine bysubstituting glutamine and asparagine or aspartic acid, respectively, atthe 26^(th) and 34^(th) positions in the peptide comprising the GLP-1(7-36) or its amino acid sequence in which one or a few amino acids aredeleted, substituted and/or added and having GLP-1 activity, throughwhich the GLP-1 derivative maintained insulin secretion promotingactivity at the same level as native GLP-1 and was given a resistance tothe digestive enzyme such as trypsin. Further, it was modified bysubstituting serine or glycine for alanine at the 8^(th) position so asto obtain the resistance to dipeptidylpeptidase IV as well to be stablealso in blood plasma.

That is, the GLP-1 derivative of the present invention is a peptidehaving an amino acid sequence in which glutamine and asparagine oraspartic acid, respectively, are substituted at the 26^(th) and 34^(th)positions in the peptide comprising the GLP-1 (7-36) or its sequence inwhich one or a few amino acids are deleted, substituted and/or added andhaving GLP-1 activity. Here the peptide comprising the GLP-1 (7-36) orits sequence in which one or a few amino acids are deleted, substitutedand/or added and having GLP-1 activity includes a precursor and ananalogue of the GLP-1 and the C-terminal amide bodies, and it ispreferably the GLP-1 (7-36), the GLP-1 (7-37) or the C-terminal amide ofthe GLP-1 (7-36) or the GLP-1 (7-37). It is particularly preferable tosubstitute serine or glycine at the 8^(th) position in the GLP-1derivative of the present invention. Dipeptidylpeptidase IV is an enzymewhich recognizes proline or alanine at the second site from theN-terminal of the polypeptide chain and hydrolyzes the carboxyl groupside. Therefore, it is preferable to substitute serine or glycine foralanine at the 8^(th) position in the GLP-1 derivative of the presentinvention. This derivative substitution at the 8^(th) position maintainsactivity at the same level as that of the native GLP-1, and is stablealso in blood plasma.

As stated above, the GLP-1 (7-36) used in the present invention is apeptide comprising the following amino acid sequence:His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg(SEQ ID NO: 7), here [Ser⁸] showing that the 8^(th) position is modifiedby serine, which indicates that the second (corresponding to the 8^(th)position) Ala is replaced with Ser. The GLP-1 derivative of the presentinvention can be produced using chemosynthesis or genetic engineeringtechniques.

That is, the principal of chemosynthesis of polypeptides is commonlyknown in the art, and a general text of the art as following can bereferred to: Dugas H. and Penny C. Bioorganic Chemistry, 1981,Springer-Verlag, New York, pp. 54-92, Merrifields J M, Chem. Soc, 85,2149, 1962, Stewart and Young, Solid Phase Peptide Synthesis, pp. 24-66,Freeman (San Francisco, 1969). The peptide of the present invention canbe synthesized by solid phase methods with the use of, e.g., 430Apeptide synthesizer (PE-Applied Biosystems Inc, 850 Lincoln CenterDrive, Foster City Calif. 94404) and synthesis cycle supplied byPE-Applied Biosystems. Boc amino acids and other reagents can bepurchased from PE-Applied Biosystems and other pharmaceutical suppliers.

Production of the GLP-1 derivative of the present invention by geneticengineering techniques can also be performed with the use of the geneobtained from total synthesis of the DNA of the GLP-1 derivative ormodification of DNA encoded by larger natural glucagons. The method forconstructing a synthetic gene is widely known in the art, and Methods inEnzymology, Academic Press, NY, vol. 68, 109-151 by Brown et al. can bereferred to.

Further, DNA used for generating the GLP-1 derivative of the presentinvention can be designed to enhance the amount of expression andaccumulate the product stably in the host, to facilitate thepurification after production, or to produce the product as a fusionprotein and cleave the GLP-1 derivative out easily, other than theabove-mentioned devices. For instance, to join it to a gene of a proteinsuch as β-galactosidase, β-lactamase, a protein A, or TrpE to generateit as a fusion protein is one of these procedures. In these cases, inorder to obtain the GLP-1 derivative as a simple substance aftergeneration, a gene corresponding to the amino acid methionine can beinserted between each gene and treated with cyanogen bromide. Here, theC-terminal is changed to Hse (homoserine). Some of the GLP-1 derivativesof the present invention have arginine only at C-terminal, so a simplesubstance of the GLP-1 derivative can be obtained by enzymatic treatmentwith an arginyl endopeptidase.

Meanwhile, the gene encoding the above GLP-1 derivative can also producethe GLP-1 derivative by being introduced into cells other than plantsand being expressed according to known genetic engineering techniques.In this case, the gene encoding the GLP-1 derivative is introduced intoa suitable recombinant DNA expression vector by using a suitablerestriction endonuclease. After constructing an expression vector forthe GLP-1 derivative, a suitable host cell is transformed by using thevector. Either eukaryotic cells or prokaryotic cells can be used as hostcells. The techniques to construct a vector and to transform cells arecommonly known in the art, and Molecular Cloning; A Laboratory Manual,Cold Springs Harbor Laboratory Press, NY. vols. 1-3, 1989 by Maniatis etal. can be generally referred to. In such case, in order to achieveefficient transcription of the subject gene, the subject gene is boundto the promoter-operator region functionally. Various expression vectorswhich can be used for transformation of eukaryotic cells or prokaryoticcells are commonly known and The Promega Biological Research ProductsCatalogue and The Stratagene Cloning Systems Catalogue can be referredto. In production of the GLP-1 derivative of the present invention,widely used substance production systems using microorganisms andmammalian culture cells as hosts can be used. Further as a stablesubstance production system at low cost, a substance production systemusing transformed plants as described above can also be used.

[Use of the GLP-1 Derivative of the Present Invention]

The GLP-1 derivative produced in the present invention can be used bytaking in the form of a storage organ such as plant seeds, in the formof a preparation by purification and isolation, or in the form of foodor drink or the like to which the constituent is added. When it is usedin the form of a preparation, it can also be used as a pharmaceutical bycombining the constituent comprising the GLP-1 derivative and apharmaceutically acceptable carrier, diluent, excipient or an absorptionpromoter formulated for pharmaceuticals. The GLP-1 derivative of thepresent invention is effective for various diseases in which the GLP-1is involved, so it can be used, for e.g., treatment ofinsulin-independent chronic diabetes mellitus, treatment forinsulin-dependent chronic diabetes mellitus, treatment for obesity, orappetite suppression.

EXAMPLES

The present invention is described below more specifically withreference to Examples, however, the present invention is not limited tothe following Examples. Meanwhile, in the following Examples, furtherdetailed experimental operations were performed according to theprocedures of molecular biology by Molecular Cloning (Sambrook et al.,1989) or the operation manual by the manufacturer unless otherwisestated.

Example 1

I. Preparation of Plasmid pGlbGLP130Hm

Rice globulin promoter cleaved using restriction enzymes EcoRI and Sse83871, rice globulin gene wherein a gene encoding [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36 amide) shown in SEQ ID NO:1 was inserted into thevariable region (the 109^(th) amino acid position), and a gene fragmentlinked to the polyadenylation signal of nopaline synthase were insertedinto the EcoRI-Sse83871 restriction enzyme site of pTL7 (H. Ebinuma etal., Molecular Methods of Plant Analysis, 22:95, 2002), to obtainplasmid pGlbGLP. As shown in SEQ ID NO:2, [Ser⁸, Gln²⁸, Asp³⁴]-GLP-1(7-36) comprises amino acids 7-36 of GLP-1, and is a derivative whereinthe 8^(th), 26^(th) and 34^(th) positions are replaced with serine,glutamine and asparagine, respectively. For insertion into the riceglobulin gene, lysine residue (AAG) was added to its N-terminal.

On the other hand, plasmid pUC18ΔKpnI was obtained by cleaving therestriction enzyme site KpnI of plasmid pUC18 with restriction enzymeKpnI, blunting its cleavage end with T4 polymerase, and then re-joined.The region between recombinant sequences Rs of the yeast site-specificrecombination system was cleaved from plasmid pNPI130 (JapaneseLaid-Open Patent Application No. 9-154580) with restriction enzymeSse8387I and inserted into the restriction enzyme site Sse8387I of thepUC18ΔKpnI, to obtain plasmid pNPI130PUC.

Further, a gene fragment linked to the CaMV35S promoter, the Hm(hygromycin-resistant) gene and the polyadenylation signal of nopalinesynthase was cleaved from the plasmid pNPI140 (Japanese Laid-Open PatentApplication No. 9-154580) with restriction enzyme KpnI, and insertedinto the restriction enzyme site KpnI of pNPI130PUC, to obtain plasmidpNPI130Hm.

The intended plasmid was obtained by cleaving from the pNPI130Hm theregion between recombinant sequences Rs of the yeast site-specificrecombination system with the restriction enzyme Sse8387I, andintroducing it between the restriction enzymes site Sse8387I of pGlbGLP,and naming it as plasmid pGlbGLP130Hm (International Accession No. FERMBP-8343). In the pGlbGLP130Hm, a gene encoding [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36) exists because it has been inserted into thevariable region (the 109^(th) amino acid position) of rice globulingene, as a recombinant protein gene allowing to express in plant storageorgan. Moreover, it comprises an ipt gene as a cytokinin-related geneand a hygromycin-resistant gene as a drug-resistant gene, and uses theyeast site-specific recombination system R/Rs system as a removable DNAelement.

The schemes for preparing pGlbGLP130Hm are shown in FIGS. 1-4, and arestriction map of the region (T-DNA region) in pGlbGLP130Hm to beintegrated into plant chromosome is shown in FIG. 4. In FIGS. 1-4, Glb-Prepresents a promoter of the globulin gene; GLP represents a geneencoding [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36); globulin represents the riceglobulin gene; T represents the polyadenylation signal of the nopalinesynthase gene; 1a represents fragment of lacZ′ gene; 35S-P representsthe 35S promoter of cauliflower mosaic virus; ipt represents an iptgene; circled-T represents the polyadenylation signal of the ipt geneitself; Hm represents hygromycin resistant gene; R representsrecombinant enzyme gene; triangle framed with a rectangle represents therecombinant sequence Rs and its sequence direction; and RB and LBrepresent boundary sequences of the T-DNA region.

II. Introduction of pGlbGLP130Hm into Agrobacterium

A. tumefaciens EHA 105 strain was inoculated in 10 mL YEB liquid medium(beef extract 5 g/L, yeast extract 1 g/L, peptone 5 g/L, sucrose 5 g/L,2 mM of MgSO₄, pH 7.2 at 22° C. (hereinafter, pH will be the value at22° C., unless otherwise stated)), and cultured at 28° C. until OD630value reached 0.4 to 0.6. The culture liquid was centrifuged at 6900×gat 4° C. for 10 min, and the bacteria harvested. Then, the bacteria wassuspended in 20 ml of 10 mM HEPES (pH 8.0), centrifuged again at 6900×gat 4° C. for 10 min and harvested. The resultant bacteria were furthersuspended in 200 μl of YEB liquid medium, and this was used as thebacterial culture for plasmid introduction.

By using the bacterial culture for plasmid introduction, theintroduction of pG1bGLP130Hm into Agrobacterium was performed asfollows. In other words, electroporation was performed with the mixedsolution of 50 μl of the above bacterial culture for plasmidintroduction and 3 μl of pGlbGLP130Hm with gene pulser II system(BIORAD) in a 0.5 ml-tube. Then, 200 μl of YEB liquid medium was addedto the resultant mixed solution after electroporation treatment, and themixture was cultured by shaking at 25° C. for 1 hour. The bacteria werefurther inoculated in YEB agar medium (agar 1.5 w/v %, other componentswere same as above) supplemented with 50 mg/L kanamycin and cultured at28° C. for 2 days. Further, the obtained bacteria colony wastransplanted into YEB liquid medium and cultured. Then plasmid wasextracted from the bacteria by alkaline method, to confirm thesebacteria were EHA 105 strain introduced with pGlbGLP130Hm, and thesewere named as EHA 105 (pGlbGLP130Hm).

III. Preparation of Infection Material

As the target of gene introduction, Oryza sativa variety “NIPPONBARE”was used, and sterilization of the ripe seeds was performed according tothe method of “Cell Engineering Annex, Plant Cell Engineering Series 4,Experiment Protocol of Model Plant (pp. 93-98)”. The sterilized ripeseeds were placed in N6C12 medium (N6 inorganic salts and vitamins, 30g/L sucrose, 2.8 g/Lproline, 0.3 g/L casamino acid, 1 mg/L 2,4-D, 4g/LGelLight, pH=5.8), sealed with surgical tape, and cultured in alighted place at 28° C. for germination, to produce a material forinfection by Agrobacterium EHA 105 (pGlbGLP130Hm).

IV. Transformation of Rice by EHA 105 (pGlbGLP130Hm) and Preparation ofTransformed Rice

Agrobacterium EHA 105 (pGlGLP130Hm) cultured in YEB agar medium (beefextract 5 g/L, yeast extract 1 g/L, peptone 5 g/L, sucrose 5 g/L, 2 mMMgSO₄, 15 g/L Bacto Agar) was transplanted into YEB liquid medium, andcultured at 25° C. at 180 rpm overnight, then centrifuged at 3000 rpmfor 20 min, and the bacteria harvested. The resultant bacteria weresuspended in N6 liquid medium (N6 inorganic salts and vitamins, 30 g/Lsucrose, 2 mg/L 2,4-D, pH=5.8) containing acetosyringone (10 mg/L) sothat OD₆₃₀=0.15, to produce an Agrobacterium suspension for infection.

Germinated seeds prepared in III were placed in a 50 ml-tube, and theabove Agrobacterium suspension for infection was added to the tube toimmerse the seeds into it. After 1.5 min of immersion, the Agrobacteriumsuspension was discarded, the germinated seeds were placed on asterilized paper filter to remove extra water, placed into N6C12 medium(N6 inorganic salts and vitamins, 30 g/L sucrose, 2.8 g/L proline, 0.3g/L casamino acid, 1 mg/L 2,4-D, 4 g/L GelLight, pH=5.2), sealed withsurgical tape, and cocultured at 28° C. in the dark for 3 days. Then,the resultant seeds were transplanted into N6C12TCH25 medium (N6inorganic salts and vitamins, 30 g/L sucrose, 2.8 g/L proline, 0.3 g/Lcasamino acid, 2 mg/L 2,4-D, 500 mg/L carbenicillin, 25 mg/L hygromycin,4 g/L GelLight) and cultured for 1 week. Then, the germinated bud wascut from the scutellum tissue of the germinated seed.

Next, the scutellum tissue was cultured in N6C14TCH25 medium (N6inorganic salts and vitamins, 30 g/L sucrose, 2.8 g/L proline, 0.3 g/Lcasamino acid, 4 mg/L 2,4-D, 500 mg/L carbenicillin, 25 mg/L hygromycin,4 g/L GelLight) for 1 week, and further cultured in MSRC medium (MSinorganic salts and vitamins, 30 g/L sucrose, 30 g/L sorbitole, 2 g/Lcasamino acid, 500 mg/L carbenicillin, 4 g/L GelLight). The bud or theseedling plant was redifferentiated during the 1^(st) to 2^(nd) monthafter coculture with EHA 105 (pGlbGLP130Hm). The redifferentiated bud orseedling plant was transplanted to rooting medium and grown, and aplantlet of about 20 cm high was obtained. Chromosomal DNA was extractedfrom the seedling plants with the use of DNeasy 96 Plant Kit (QIAGEN),and the existence of the gene encoding [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36)was confirmed by PCR method.

At that time, as PCR primer to detect the gene encoding [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36) introduced into the variable region of globulingene, primers 3-1:5′-GGATCCATGGCTAGCAAGGTCGTC-3′ (SEQ ID NO: 3) and3-3:5′-GATCACTATCTCGTTGCATGCAACAC-3′ (SEQ ID NO:4) were used. Theobtained PCR reactant (about 700 bp) was analyzed by agarose gelelectrophoresis and the existence of the gene encoding [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36) in the chromosomal DNA was confirmed.

As a result, it was revealed that the above gene encoding [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36) was introduced into about 3% of the Oryza sativaseeds provided for Agrobacterium infection treatment.

The transformants of the plantlets of Oryza sativa confirmed to beintroduced with the gene encoding [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) thusobtained, were transferred to soil and grown to harvest ripe seeds in aroom where sunlight enters.

V. Protein Analysis

10 mg of ripe seeds obtained in IV. was treated with 250 μl of 62.5 mMTris-HCl (pH 6.8) extract buffer containing 10% (v/v) glycerol, 0.25%(w/v) SDS, 5% 2-mercapto ethanol, at 100° C. for 5 min to extract allproteins of these seeds. The extract solution was provided for analysisby SDS-PAGE. For SDS-PAGE, 15% (w/v) polyacrylamide (acrylamide:N,N′-methylenebisacrylamide=30:0.8) gel was used.

The obtained gel image was analyzed with an analysis software, ImageGauge (Fujifilm), and the accumulation level of fusion protein whereinthe gene encoding [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) was inserted intothe variable region of globulin was examined. The results are shown inFIG. 5.

Comparative Example 1

Except for performing gene introduction into ripe Oryza sativa seeds byusing the conventional vector pGlbGLP-Hm shown in FIG. 6 comprising riceglobulin promoter, a globulin gene wherein the gene encoding [Ser⁸,Gln²⁶, Asp³⁴]-GLP-1 (7-36) shown in SEQ ID NO: 1 was inserted into thevariable region, and a gene fragment linked to the polyadenylationsignal of nopaline synthase, the gene introduction was performed in thesame manner as Example 1 to redifferentiate the bud or the seedlingplant. By regenerating a plant from the bud or the seedling plant, atransformed Oryza sativa was obtained and ripe seeds were collected. Theripe seeds were submitted to protein analysis. The results are shown inFIG. 5.

As it is clear from FIG. 5, the fusion protein wherein the gene encoding[Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) is inserted into the variable regionof globulin was highly accumulated in the rice ripe seeds produced inExample 1, and showed about 6-fold level at maximum, compared withComparative Example 1 (FIG. 5).

Example 2 I. Synthesis of GLP-1 Derivative

The GLP-1 derivatives shown in the following were synthesized by solidphase synthesis using Model 430A peptide synthesizer (PE-AppliedBiosystems, Foster City, Calif.), purified by HPLC. The synthesizedmaterials were confirmed by mass spectrum. Derivatives with 95% or morepurity were used for in vitro and in vivo examination.

Comparative Production Example 1. GLP-1 (7-36 amide) (Native GLP-1)Comparative Production Example 2. [Ser⁸]-GLP-1 (7-36 amide)Comparative Production Example 3. [Gly⁸]-GLP-1 (7-36 amide)Production Example 1. [Gln²⁶, Asn³⁴]-GLP-1 (7-36 amide)(The amino acid sequence of non-amide body of Production Example 1 isshown in SEQ ID NO:5).

Production Example 2. [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) ProductionExample 3. [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36)

(The amino acid sequence of Production Example 3 is shown in SEQ IDNO:6).

II. Cyclic AMP Production Activity of the GLP-1 Derivative

Expression vectors were constructed according to the published DNAsequence (Graziano et al, Biochem Biophys Res Com, 196:141-146, 1993) ofthe human GlP-1 receptor. Chinese hamster ovary CHO-K1 cells weretransformed with the vectors, and the recombinant CHO-K1 cells whichexpress human GLP-1 receptors were obtained. The human GLP-1receptor-expression cells were placed in 24-well plates at 1×10⁴cells/ml/well, and were used for assay 3 days later.

The assay was performed as follows: the cells were incubated in thepresence of the GLP-1 derivatives in a buffer (PBS, 5.6 mM glucose, 1 mMisobutyl methyl xanthine, 20 μM Ro20-1724, 0.5% BSA, pH 7.4) at 37° C.for 30 min. 10 μl of 5N hydrochloric acid was added to the buffer tostop the incubation. Cyclic AMP formed in the cells by the reaction ofvarious GLP-1 derivatives and GLP-1 receptors was measured by enzymeimmunoassay with cAMP-Screen™ system (Applied Biosystems). FIG. 7 showscyclic AMP production activity of various GLP-1 derivatives.

As a result, [Ser⁸]-GLP-1 (7-36 amide), [Gly⁸]-GLP-1 (7-36 amide) and[Gln²⁶, Asn³⁴]-GLP-1 (7-36 amide) had the same level of cyclic AMPproduction activity as native GLP-1.

Example 3

Trypsin resistance of [Gln²⁶, Asn³⁴]-GLP-1 (7-36 amide) of ProductionExample 1 was examined by measuring cyclic AMP production activity inthe same manner as Example 2, after trypsin treatment.

In other words, the above GLP-1 derivative obtained by synthesis wasdissolved in 50 mM ammonium hydrogen carbonate solution (pH 7.8), sothat the concentration becomes 500 μg/mg. 5 μl of 500 μg/ml trypsinsolution (Promega: Cat. No. V5113) was added to 100 μl of this solution,and reacted at 37° C. for 1 hour. The reaction was stopped by adding1200 μl of 71.5% ethanol (final 65%). The supernatant was collected bycentrifugation at 15,000 rpm at 4° C. for 5 min, and evaporation wascarried out. The dried solids were dissolved in distilled water and usedfor measuring activity.

FIG. 8 shows the concentration dependency of [Gln²⁶, Asn³⁴]-GLP-1 (7-36amide) activity before and after trypsin treatment. [Gln²⁶, Asn³⁴]-GLP-1(7-36 amide) showed no difference of activity before and after trypsintreatment, and it was revealed that it was resistant to trypsin.

Example 4

Stability to Pepsin of the GLP-1 Derivative in Ripe Seeds of Oryzasativa was Examined

Polished rice and powder thereof of the ripe seeds of Oryza sativaobtained in Example 1 were used and cooked with 1.9-fold amount of waterat 100° C. by heating for 15 min. Those in the form of granules werecrushed and homogenized, diluted 5-fold with distilled water to make asample. Those in powder were directly diluted 5-fold with distilledwater to make a sample. On the other hand, as for synthetic GLP-1 (7-36amide), [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1(7-36), a 10 μg/ml solution was prepared with 0.2% BSA solution as asample.

A 1/10-amount of artificial gastric juice (pH1.2) of 10-foldconcentration containing 7.6 mg/ml pepsin was added to each sample, andthe liquid was neutralized with NaOH after reaction at 37° C. for 1hour. Then, as for GLP-1 (7-36 amide), [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36)and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36), after extracting the protein andgetting GLP-1 simple substance by trypsin treatment as for those derivedfrom rice, the activity according to cyclic AMP production was measured.As a result, it was revealed that the activity of synthetic GLP-1derivative was completely lost by pepsin treatment, while 31-65% ofGLP-1 activity remained in rice (FIG. 9).

From these results, it can be estimated that GLP-1 derivatives containedin rice ripe seeds are not easily digested by pepsin and can reach thesmall intestine by passing through the stomach.

Example 5

The fusion protein with [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) and globulinwas extracted from ripe seeds of Oryza sativa obtained in Example 1 with0.025 M sodium hydroxide solution, and the extract was diluted 15-foldwith 50 mM ammonium hydrogen carbonate pH 7.8. To this diluent, 6 μl of83 μg/ml trypsin solution (Promega: Cat. No. V5113) was added. Theresultant was reacted at 37° C. for 1, 2, 4, 6 or 20 hours, and then thereaction was stopped by adding 1200 μl of 71.5% ethanol (final 65%). Thesupernatant was collected by centrifugation at 15,000 rpm at 4° C. for 5min and evaporation was carried out. The dried solids were dissolved indistilled water and the activity was measured.

FIG. 10 shows the relationship between trypsin treatment time andactivity of [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) which has been expressedin ripe seeds of Oryza sativa and obtained as fusion protein. Cyclic AMPproduction activity appears only by trypsin treatment, and the activitywas maintained regardless of the trypsin treatment time. From theseresults, it was revealed that [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) isexpressed in ripe seeds of Oryza sativa as a form having the activityand trypsin-resistance. Therefore, it is estimated that [Ser⁸, Gln²⁶,Asp³⁴]-GLP-1 (7-36) contained in ripe seeds of Oryza sativa by the GLP-1derivative expression can be absorbed without being degraded by trypsinin the small intestine.

Example 6

Trypsin-resistance of [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) and [Ser⁸,Gln²⁶, Asp³⁴]-GLP-1 (7-36) was examined by measuring cyclic AMPproduction activity in the same manner as Example 2 after trypsintreatment.

In other words, 8 μl of the above synthetic GLP-1 derivative, diluted to10 μg/ml with 0.2% bovine serum albumin solution was added to 112 μl of50 mM ammonium hydrogen carbonate (pH 7.8) and 6 μl of 83 μg/ml trypsinsolution (Promega:Cat. No. V5113) for reaction at 37° C. for 1 hour. Thereaction was stopped by adding 1200 μl of 71.5% ethanol (final 65%). Thesupernatant was collected by centrifugation of 15,000 rpm at 4° C. for 5min and evaporation was carried out. The dried solids were dissolved indistilled water and used for measuring the activity.

FIG. 11 shows the variation in activity against trypsin treatment timeof GLP-1 (7-36 amide), [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) and [Ser⁸,Gln²⁶, Asp³⁴]-GLP-1 (7-36). Compared with native GLP-1 (7-36 amide),[Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) and [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36)showed no variation inactivity by trypsin treatment, and they wererevealed to be trypsin resistant.

Example 7

It was examined whether the GLP-1 derivative of the present inventionshows significant DDP-IV resistance compared with native GLP-1. 5000 pMof GLP-1 (7-36 amide) (native GLP-1), 500 pM of [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1 (7-36), and 5000 pM of [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36)were mixed separately with 40 μU/μl of DPP-IV (Sigma, D7052) forreaction at 37° C. for 0, 15, 30 and 60 min, the mixtures were extractedwith 2-fold amount of ethanol and the extracts were dried withcentrifugal evaporator. The obtained dried solids were dissolved indistilled water containing 1% BSA and were reacted with GLP-1receptor-expression cells to measure the cyclic AMP production level.FIG. 12 shows the comparison of cyclic AMP production activity with 100%for those without DPP-IV treatment. Compared to GLP-1 (7-36 amide),[Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) and [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36)showed obvious DPP-IV resistance.

Example 8

Insulin-secretion-promoting activity of GLP-1 derivative of the presentinvention was examined. Langerhans islets were extracted from ICR mousepancreas with collagenase, 2 to 3 Langerhans islets were placed per wellof 24-well plates, and cultured overnight. Then, the GLP-1 derivative ofthe present invention dissolved in Krebs-Ringer buffer containing 16.7mM glucose, 0.2% BSA and 10 mM hepes was added, incubated at 37° C. for30 min and insulin concentration in the supernatant was measured with anenzyme immunoassay kit (Shibayagi).

Amount-dependent insulin secretion-promoting activity was observed ineach of the peptides of GLP-1 (7-36 amide), [Ser⁸, Gln²⁶, Asp³⁴]-GLP-1(7-36) and [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36). Particularly, a stronginsulin secretion-promoting activity was observed in [Ser⁸, Gln²⁶,Asn³⁴]-GLP-1 (7-36) at high concentration (FIG. 13).

Example 9

Hypoglycemic effect in oral glucose tolerance test (OGTT) by the GLP-1derivative administered subcutaneously was examined.

1 g/kg glucose was orally administered to mice fasted overnight, andimmediately, GLP-1 (7-36 amide), [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) or[Ser⁸, Gln²⁶, Asp³⁴]-GLP-1 (7-36) was administered by dorsalsubcutaneous administration (5, 20 μg/kg). Physiological saline wasadministered to the control group. Before loading glucose and 20, 60,120 min after loading, blood was collected chronologically fromsubocular venous plexus, to measure blood glucose level. In the GLP-1derivative, it was observed that the peak value of blood glucoseincrease has a tendency to decrease, and a strong action was confirmedin [Ser⁸, Gln²⁶, Asn³⁴]-GLP-1 (7-36) (FIG. 14). Moreover, the actioncontinued until 120 min after the administration (FIG. 15). It wasrevealed that by the modification of the GLP-1 peptide, the stability inblood in vivo significantly increased and the sustainability wasassured.

INDUSTRIAL APPLICABILITY

The present invention provides a substance production system by a methodfor producing a plant storage organ in which a recombinant protein ishighly produced as a safe and an efficient substance production systemat low cost with the use of genetic engineering. The method of thepresent invention can provide food in which an ingredient useful forpromoting health is significantly accumulated. Further, the method ofthe present invention can be used as a basic technology to generatehigh-value added plant which produces valuable substance aspharmaceuticals or industrial materials.

Further, the present invention encompasses the production of a GLP-1which is known as a hormone secreted from the digestive tract by foodintake and acting on the pancreas to stimulate glucose-dependent insulinsecretion, according to the method of the present invention.

In addition, the novel GLP-1 derivative provided in the presentinvention has excellent properties: it is resistant to a digestiveenzyme such as trypsin which causes a problem when GLP-1 is used, and itfurther has resistance to dipeptidylpeptidase IV which causes a problemwith stability in blood plasma after it is taken and absorbed, thereforeit can be expected for use as a pharmaceutical. That is, it is possiblefor the GLP-1 derivative of the present invention to express itstherapeutic effect even when it is orally taken, and for instance, evenwhen it is expressed in a plant storage organ by the method of thepresent invention and orally taken, it can be absorbed from the smallintestine without being degraded and express its therapeutic effect.Accordingly, as the GLP-1 derivative provided by the present inventionenhances the possibility of the clinical application of GLP-1, and it isbelieved that it helps improve the quality of life of diabetic patientsand obese patients.

1-18. (canceled)
 19. A method for producing a plant storage organ inwhich a GLP-1 derivative recombinant protein is highly produced, whereina gene encoding the GLP-1 derivative in which glutamine is substitutedat the 26th position and asparagine or asparatic acid is substituted atthe 34th position in a peptide comprising GLP-1 (7-36) or its amino acidsequence in which one or a few amino acids are deleted, substitutedand/or added, and having a GLP-1 activity is used as a recombinantprotein gene to be expressed in a plant storage organ, and which methodcomprises the following steps (A), (B), and (C): (A) a step ofconstructing a vector which comprises a recombinant protein gene to beexpressed in a plant storage organ, a cytokinin-related gene, adrug-resistant gene, and a removable DNA element, wherein thecytokinin-related gene and the drug-resistant gene exist in thepositions so that they can behave together with the removable DNAelement, while the recombinant protein gene to be expressed in the plantstorage organ exists in the position so that it would not behavetogether with the removable DNA element, and introducing the vector intocells, (B) a step of redifferentiating transformant by culturing theplant cell into which the vector is introduced by said step (A) in adrug-supplemented medium and a drug-free medium, and (C) a step ofobtaining the plant storage organ from the transformant redifferentiatedin said step (B).
 20. The method for producing a plant storage organ inwhich a GLP-1 derivative recombinant protein is highly producedaccording to claim 19, wherein the gene encoding GLP-1 derivative is agene encoding GLP-1 derivative in which serine or glycine is furthersubstituted at the 8th position in the amino acid sequence.
 21. Themethod for producing a plant storage organ in which a GLP-1 derivativerecombinant protein is highly produced according to claim 20, whereinthe gene encoding GLP-1 derivative is a gene shown in SEQ ID No:1 of thesequence listing.
 22. The method for producing a plant storage organ inwhich a GLP-1 derivative recombinant protein is highly producedaccording to claim 19, wherein the GLP-1 derivative consists of theamino acid sequence shown in SEQ ID No: 2, 5 or 6 of the sequencelisting.
 23. The method for producing a plant storage organ in which aGLP-1 derivative recombinant protein is highly produced according toclaim 19, comprising culturing the plant cell into which the vector isintroduced in a plant hormone-free medium and drug-free medium afterculturing it in a plant hormone-supplemented medium anddrug-supplemented medium, during the step of redifferentiatingtransformant from the plant cell into which the vector has beenintroduced.
 24. The method for producing a plant storage organ in whicha GLP-1 derivative recombinant protein is highly produced according toclaim 19, wherein the gene encoding GLP-1 derivative is under control ofa promoter specific to the plant storage organ.
 25. The method forproducing a plant storage organ in which a GLP-1 derivative recombinantprotein is highly produced according to claim 19, wherein the geneencoding GLP-1 derivative is inserted into or is substituted for thesite encoding protein variable region, in the gene encoding the proteinoriginally expressed in the plant storage organ.
 26. The method forproducing a plant storage organ in which a GLP-1 derivative recombinantprotein is highly produced according to claim 25, wherein a nucleotidesequence which encodes an amino acid sequence for enzyme cleavage tocleave and separate the GLP-1 derivative from the protein originallyexpressed in the plant storage organ is placed into the boundary betweenthe gene encoding GLP-1 derivative and the gene encoding the proteinoriginally expressed in the plant storage organ.
 27. The method forproducing a plant storage organ in which a GLP-1 derivative recombinantprotein is highly produced according to claim 19, wherein the plantstorage organ is a seed.
 28. The method for producing a plant storageorgan in which a GLP-1 derivative recombinant protein is highly producedaccording to claim 27, wherein the gene encoding the protein originallyexpressed in the plant storage organ to be inserted into or to besubstituted for the protein variable region is a seed storage proteingene.
 29. The method for producing a plant storage organ in which aGLP-1 derivative recombinant protein is highly produced according toclaim 19, wherein the cytokinin-related gene is a cytokinin-synthesisgene.
 30. The method for producing a plant storage organ in which aGLP-1 derivative recombinant protein is highly produced according toclaim 29, wherein the cytokinin-synthesis gene is an isopentenyltransferase gene.
 31. The method for producing a plant storage organ inwhich a GLP-1 derivative recombinant protein is highly producedaccording to claim 19, wherein the drug-resistant gene is ahygromycin-resistant gene.
 32. The method for producing a plant storageorgan in which a GLP-1 derivative recombinant protein is highly producedaccording to claim 19, wherein the removable DNA element is derived froma site-specific recombination system or a transposon.
 33. The method forproducing a plant storage organ in which a GLP-1 derivative recombinantprotein is highly produced according to claim 19, wherein the plant ismonocotyledon.
 34. The method for producing a plant storage organ inwhich a GLP-1 derivative recombinant protein is highly producedaccording to claim 33, wherein the monocotyledon is Oryza sativa.
 35. Aplant storage organ in which a GLP-1 derivative recombinant protein ishighly produced by the method for producing according to claim 19, or atransformed plant to produce the plant storage organ.
 36. A recombinantvector for introducing a gene into a plant to use in the method forproducing a plant storage organ in which a GLP-1 derivative recombinantprotein is highly produced according to claim 19, wherein a geneencoding the GLP-1 derivative in which glutamine is substituted at the26th position and asparagine or asparatic acid is substituted at the34th position in a peptide comprising GLP-1 (7-36) or its amino acidsequence in which one or a few amino acids are deleted, substitutedand/or added, and having a GLP-1 activity is used as a recombinantprotein gene to be expressed in a plant storage organ; and whichrecombinant vector comprises a gene encoding the GLP-1 derivative; acytokinin-related gene, a drug-resistant gene and a removable DNAelement; and which gene has been introduced into a vector wherein thecytokinin-related gene and the drug-resistant gene exist in thepositions so that they can behave together with the removable DNAelement, while the recombinant protein gene to be expressed in the plantstorage organ exists in the position so that it would not behavetogether with the removable DNA element.
 37. The vector for geneintroduction into a plant according to claim 36, wherein the geneencoding GLP-1 derivative is a gene encoding a peptide in which serineor glycine is further substituted at the 8th position in the amino acidsequence.
 38. The vector for gene introduction into a plant according toclaim 37, wherein the gene encoding GLP-1 derivative is a gene shown inSEQ ID No:1 of the sequence listing.